PROJECT 3: Hormonal influences on insect motoneurons during post- embryonic development. Steroid hormones exert powerful influences on neural circuits and behavior through their actions on the CNS. Identifying the cellular targets of these hormones and the molecular mechanisms through which they act are essential for understanding how the CNS is modified post-embryonically to accommodate normal neural function, these efforts are often hindered by the cellular heterogeneity and complex interactions that characterize the CNS. In the proposed experiments we will exploit two well characterized insect model systems that offer complementary advances. During metamorphosis in the moth, Manduca, changes in their synaptic inputs and target muscles. We have shown, using primary cell culture, that the steroid hormone, 20-hydroxyecdysone (20E) acts directly on the motoneurons of the appropriate stage to regulate neurite outgrowth and branching, as well as the levels of voltage- gated Ca2+ currents. The principal goals of the proposed experiments are to characterize the intracellular mechanisms of steroid hormone action and the relative influences of hormonal vs. inter-cellular interactions in regulating this remodeling. Whereas Manduca offers the advantages of large size for manipulation and electrophysiology and well-characterized endocrine titers. Drosophila, which undergoes similar motoneuron remodeling, offers powerful molecular-genetic approaches to exploring signaling pathways. In the first specific aim a new organ culture protocol and cell imaging system will be used to describe the dendritic modeling of identified flight motoneurons in Manduca and Drosophila in their natural context, in vivo, and to determine the roles of specific synaptic inputs and 20E. In the second specific aim, the intracellular 20E response pathway will be examined by describing the temporal expression patterns of ecdysteroid receptors and a key primary response key BRC in identified motoneurons as they are being remodeled. Steroid hormone regulation of key elements in the response pathway will be examined in primary cell culture. The requirement for specific primary response genes will be determined by examining appropriate Drosophila mutants, in collaboration with investigators of project 4. The third specific aim follows from our observation that 20 E affects both Ca2+ currents and the duration of internal Ca2+ increases, and that changes in Ca2+ current levels, in vivo, Ca2+ levels, Ca2+ release from internal stores, and the Ca2+-dependent enzyme, CAM-kinase II, in ecdysteroid-induced dendritic remodeling using voltage clamp, Ca2+ imaging and genetic approaches. The basic mechanisms revealed by these studies will contribute to our understanding of CNS plasticity during normal maturation, and following injury or disease.